Synthesis method and detection method of Pulsatiloside A

Abstract

The invention provides a synthetic method of Pulsatilloside A. The Pulsatilloside A is prepared by taking pulsatilla saponin B4 as a starting raw material. The invention further provides a detection method of the pulsatabiloside A. The invention further provides a detection method of the pulsatabiloside A. According to the invention, pulsatiloside A is synthesized by taking pulsatilla saponin B4 as a raw material, and the pulsatilla saponin B4 is one of the main components of the traditional Chinese medicine pulsatilla, and is rich and reliable in source. The pulsatilla saponin B4 is subjected to structural modification, separation and purification, the steps are simple and reliable, and the compound Pulsatiloside A which is rare in nature can be obtained. The invention provides the detection method of the Pulsatiloside A, the detection method is stable, and the quality control of the Pulsatiloside A can be realized; meanwhile, the pulsatilla saponin A3 and 23-hydroxybetulinic acid can be detected, and the quality control on the three components can be realized at the same time.

Description

Technical Field

[0001] The invention relates to a synthesis method and a detection method of Pulsatilloside A. Summary of the invention

[0002] Pulsatilloside A (PSA) (CAS: 136684-41-8) is a triterpenoid saponin compound extracted from the plant genus Pulsatilla. The genus Pulsatilla contains about 40 species of perennial herbs, mainly distributed in grasslands and steppes in North America, Europe and Asia. In China, Pulsatilla decoction has been used to treat diseases caused by bacteria for thousands of years, among which Pulsatilla chinensis (also known as Bai Tou Weng) is one of its main herbal sources. Studies have shown that the main active ingredients in Pulsatilla plants are saponins, such as Pulsatilloside A, Pulchinenoside C (PSC or Anemoside B4, AB4), Pulsatilla saponin D (PSD or SB365) and Pulsatilloside E (PSE). The article “TRITERPENOIDS FROMPULSATILLA CHINENSIS” by WEN-Cai Ye et al. extracted Pulsatilloside A from Pulsatilla chinensis for the first time.

[0003] Currently, Pulsatilloside A is extracted from plants, and there is no literature report on the synthesis of Pulsatilloside A. Summary of the invention

[0005] The invention provides a synthesis method and a detection method of Pulsatilloside A.

[0006] The present invention provides a method for synthesizing Pulsatilloside A, which is prepared from Pulsatilloside B4 as a starting material, and the synthetic route is:

[0007]

[0008] It includes the following steps:

[0009] a. Weigh 1 part of Pulsatilla saponin B4, add 10-20 parts of 2.5%-10% alkaline solution, heat and reflux at 50-70°C for 0.5-1.5h, add dilute sulfuric acid to adjust the pH to 6-7, and obtain Pulsatilla saponin A3; the alkaline solution is NaOH aqueous solution, KOH aqueous solution, ammonia aqueous solution;

[0010] b. Purification of Pulsatilla saponin A3;

[0011] c. Take the purified Pulsatilla saponin A3, add 10-40 times 1%-3% acid solution, heat and reflux at 50-70°C for 0.5-1.5h, add dilute sodium hydroxide solution to adjust the pH to 6-7; the acid solution is H2SO4 methanol solution, HCl methanol solution, H3PO4 methanol solution;

[0012] d. Purification: Take the mixture prepared in step c, use C-18 filler as the stationary phase, water as mobile phase A, methanol as mobile phase B, and the detection wavelength is 201 nm. Perform high pressure preparation according to the gradient in the table below, collect the sample segment, and dry it.

[0013] High pressure preparation mobile phase ratio

[0014]

[0015] Wherein, the alkaline solution described in step a is a NaOH or KOH aqueous solution with a concentration of 5%, and the material-liquid ratio is 1 part of Pulsatilla saponin B4 to 20 parts of alkaline solution; the heating reflux temperature is 60°C-70°C, and the reflux time is 1-1.5h.

[0016] The acid solution is H2SO4 methanol solution with a concentration of 2%; the material-liquid ratio is 1:20-40; the hydrolysis temperature is 60°C and the time is 0.5h.

[0017] The present invention provides a method for detecting Pulsatilloside A, which adopts HPLC detection, and the chromatographic conditions are:

[0018] Octadecylsilane bonded silica gel was used as filler (Agilent Extend-C18, 4.6 mm × 250 mm, 5 μm); water (A) and acetonitrile (B) were used as mobile phases; the detection wavelength was 201 nm; the column temperature was 30°C, the flow rate was 1.0 ml / min, and the elution conditions were shown in the following table:

[0019] Mobile phase conditions

[0020]

[0021] Specifically, it includes the following steps:

[0022] a. Preparation of reference solution:

[0023] Take Pulsatilloside A3, Pulsatilloside A, and 23-hydroxybetulinic acid reference substances, accurately weigh them, place them in a brown volumetric bottle, and add methanol to make solutions containing 0.5 mg, 0.35 mg, and 0.6 mg per 1 ml.

[0024] b. Preparation of test solution Take the Pulsatilloside A reaction solid, weigh it accurately, put it in a brown volumetric flask, add methanol to the mark, and obtain;

[0025] c. Determination method:

[0026] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into a liquid chromatograph, and measure according to the chromatographic conditions described in claim 5 to obtain the product.

[0027] The relative retention time of Pulsatilloside A is 22.63±0.52min.

[0028] The present invention uses pulsatilloside B4 as a raw material to synthesize Pulsatilloside A, which is one of the main components of the traditional Chinese medicine Pulsatilloside, and has a rich and reliable source. The present invention modifies the structure of pulsatilloside B4, separates and purifies it, and the steps are simple and reliable, so that Pulsatilloside A, a compound rarely found in nature, can be obtained. The present invention provides a detection method for Pulsatilloside A, which is stable and can achieve quality control of Pulsatilloside A; and can simultaneously detect pulsatilloside A3 and 23-hydroxybetulinic acid, so as to achieve quality control of the three components at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Pulsatilloside A H NMR, C NMR, and MS;

[0030] Figure 2 HPLC chromatogram of Pulsatilloside A detection (wherein, 1. Pulsatilloside A3;

[0031] 2. Pulsatilloside A, 3. 23-Hydroxybetulinic acid). DETAILED DESCRIPTION

[0032] Example 1 Synthesis method of Pulsatilloside A of the present invention

[0033] Take 20g of Pulsatilla saponin B4, weigh accurately, add 20 times 5% NaOH aqueous solution, heat and reflux at 60°C for 1h, then add dilute sulfuric acid to adjust the pH to 6-7 to obtain Pulsatilla saponin A3; filter, add 5 times water to wash the filter cake twice, and dry the filter cake at 60°C under reduced pressure to obtain purified Pulsatilla saponin A3;

[0034] Take 5g of Pulsatilla saponin A3, weigh accurately, add 20 times 2% H2SO4 methanol solution, heat and reflux at 60℃ for 0.5h, then add dilute sodium hydroxide to adjust the pH to 6-7;

[0035] Purification of Pulsatilloside A

[0036] Take the acid hydrolysis process verification sample, use C-18 filler as the stationary phase, water as mobile phase A, methanol as mobile phase B, the detection wavelength is 201nm, perform high pressure preparation according to the gradient in Table 1, collect the sample segment, and dry it to obtain.

[0037] Table 1 Ratio of mobile phase for high pressure preparation

[0038]

[0039] Example 2 Screening test of synthesis process parameters of the present invention

[0040] 1. Single factor test of the reaction of Pulsatilla saponin B4 to Pulsatilla saponin A3

[0041] Taking the conversion rate of Pulsatilla saponin B4 as an indicator, the single factor experimental method was used to investigate the effects of alkali type, hydrolysis temperature, alkali solution concentration, solid-liquid ratio and hydrolysis time on the conversion rate of Pulsatilla saponin B4.

[0042] Chromatographic conditions: octadecylsilane bonded silica gel as filler (Agilent Extend-C18, 4.6 mm × 250 mm, 5 μm); water (A) and acetonitrile (B) as mobile phases; detection wavelength 201 nm; column temperature 30 ° C, flow rate 1.0 ml / min. Elution conditions are shown in Table 2.

[0043] Table 2 Mobile phase conditions

[0044]

[0045] Preparation of reference solution: Weigh 5.0 mg of scutellaria saponin B4 and scutellaria saponin A3 reference substances, accurately weigh, place in a 25 ml volumetric flask, add methanol to dissolve to the scale, filter, and take the filtrate to obtain.

[0046] Preparation of test solution: Accurately measure 1.0 ml of Pulsatilla saponin reaction solution, place it in a 25 ml volumetric flask, add methanol to dissolve to the scale, filter, and take the filtrate to obtain the test solution.

[0047] Determination: Accurately aspirate 10μ1 of reference solution and sample solution and inject into liquid chromatograph for determination.

[0048] 1.1 Investigation of alkali types

[0049] Take 3 portions of Pulsatilla saponin B4, 1 g each, weigh accurately, add 20 times 5% NaOH aqueous solution, 5% KOH aqueous solution, 5% ammonia aqueous solution, heat and reflux at 60°C for 1 hour, add dilute sulfuric acid to adjust the pH to 6-7. Shake well, and test according to the content determination method. The results are shown in Table 3.

[0050] Table 3 Results of investigation on alkali types

[0051]

[0052] According to the results of the investigation on the types of alkali, the conversion rate of Pulsatilla saponin B4 was used as an indicator, and hydrolysis was carried out under different types of alkali solutions. The results showed that 5% NaOH and 5% KOH could achieve complete conversion rate of Pulsatilla saponin B4 (100%), so NaOH or KOH was selected for the experiment.

[0053] 1.2 Investigation of alkaline hydrolysis temperature

[0054] Take 3 portions of Pulsatilla saponin B4, 1 g each, weigh accurately, add 20 times 5% NaOH aqueous solution, heat and reflux at 50°C, 60°C, and 70°C for 1 hour, add dilute sulfuric acid to adjust the pH to 6-7. Shake well and test according to the content determination method. The results are shown in Table 4

[0055] Table 4 Alkaline hydrolysis temperature investigation results

[0056]

[0057]

[0058] According to the results of alkaline hydrolysis temperature investigation, the conversion rate of scutellaria baicalensis saponin B4 was used as an indicator, and hydrolysis was carried out at different temperatures. The results showed that the conversion rate of scutellaria baicalensis saponin B4 was the highest (100%) at 60-70°C, so 60-70°C was selected for the experiment.

[0059] 1.3 Investigation of alkali solution concentration

[0060] Take 3 portions of Pulsatilla saponin B4, 1 g each, weigh accurately, add 20 times 2.5% NaOH aqueous solution, 5% NaOH aqueous solution, 10% NaOH aqueous solution respectively, heat and reflux at 60°C for 1 hour, add dilute sulfuric acid to adjust the pH to 6-7. Shake well, and test according to the content determination method. The results are shown in Table 5

[0061] Table 5 Alkali solution concentration investigation results

[0062]

[0063] According to the results of the alkali solution concentration investigation, the conversion rate of scutellaria baicalensis saponin B4 was used as an indicator, and hydrolysis was carried out under different types of alkali solution concentrations. The results showed that 5% NaOH and 10% NaOH could completely convert scutellaria baicalensis saponin B4 (100%). Considering environmental factors, 5% NaOH was selected for the experiment.

[0064] 1.4 Investigation of material-liquid ratio

[0065] Take 3 parts of Pulsatilla saponin B4, accurately weigh 0.5g, 1g, and 2g respectively, add 5% NaOH aqueous solution according to the solid-liquid ratio of 1:40, 1:20, and 1:10 respectively, heat and reflux at 60℃ for 1h, add dilute sulfuric acid to adjust the pH to 6-7. Shake well and test according to the content determination method. The results are shown in Table 6

[0066] Table 6 Results of investigation on material-liquid ratio

[0067]

[0068] According to the results of the solid-liquid ratio investigation, the conversion rate of scutellaria saponin B4 was taken as an indicator, and hydrolysis was carried out according to different solid-liquid ratios. The results showed that the solid-liquid ratios of 1:40 and 1:20 could achieve complete conversion rate of scutellaria saponin B4 (100%). Considering environmental factors and reagent dosage factors, the solid-liquid ratio of 1:20 was selected for the experiment.

[0069] 1.5 Alkaline hydrolysis time investigation

[0070] Take 3 portions of Pulsatilla saponin B4, 1 g each, weigh accurately, add 20 times 5% NaOH aqueous solution, heat and reflux at 60°C for 0.5h, 1h, and 1.5h, respectively, then add dilute sulfuric acid to adjust the pH to 6-7. Shake well and test according to the content determination method. The results are shown in Table 7

[0071] Table 7 Alkaline hydrolysis time investigation results

[0072]

[0073]

[0074] According to the results of the hydrolysis time investigation, the conversion rate of scutellaria baicalensis saponin B4 was taken as an indicator, and hydrolysis was carried out according to different solid-liquid ratios. The results showed that the hydrolysis time of 1 and 1.5 h could achieve complete conversion rate of scutellaria baicalensis saponin B4 (100%), so the hydrolysis time of 1-1.5 h was selected for the experiment.

[0075] According to the results of the alkaline hydrolysis single factor test, the conditions for the reaction of scutellaria baicalensis saponin B4 to scutellaria baicalensis saponin A3 were proposed as follows: solid-liquid ratio 1:20, 5% NaOH or 5% KOH aqueous solution, heating under reflux at 60-70°C for 1-1.5h, and adjusting the pH to 6-7 with dilute sulfuric acid.

[0076] 1.6 Alkaline hydrolysis process verification

[0077] Take 3 portions of Pulsatilla saponin B4, 20 g each, weigh accurately, add 20 times 5% NaOH aqueous solution, heat and reflux at 60°C for 1 hour, then add dilute sulfuric acid to adjust the pH to 6-7. Shake well and test according to the content determination method. The results are shown in Table 8

[0078] Table 8 Alkaline hydrolysis time investigation results

[0079]

[0080] According to the process verification results, the conversion rate of scutellaria baicalensis saponin B4 was used as an indicator and hydrolysis was carried out according to the single-factor optimal conditions. The results showed that the conversion rates of the three batches of scutellaria baicalensis saponin B4 were complete (100%).

[0081] 1.7 Purification of Pulsatilla saponin A3

[0082] Take the alkaline hydrolysis process verification sample, filter it with suction, add 5 times the amount of water to the filter cake and wash it twice, and dry the filter cake under reduced pressure at 60°C to obtain it.

[0083] 2. Pulsatilloside A3 reaction is a single factor test for Pulsatilloside A

[0084] During the reaction of Pulsatilloside A3 to Pulsatilloside A, Pulsatilloside A and 23-hydroxybetulinic acid are generated simultaneously. Therefore, it is necessary to control the ratio of Pulsatilloside A to 23-hydroxybetulinic acid products to maximize the content of Pulsatilloside A in the reaction product. In this experiment, the concentrations of Pulsatilloside A and 23-hydroxybetulinic acid in the reaction product were used as evaluation indicators. According to the single-factor experimental method, the effects of acid type, hydrolysis time, hydrolysis temperature, solid-liquid ratio and acid concentration on the hydrolysis efficiency were investigated.

[0085] Chromatographic conditions: Octadecylsilane bonded silica gel as filler (Agilent Extend-C18, 4.6 mm × 250 mm, 5 μm); water (A) and acetonitrile (B) as mobile phases; detection wavelength 201 nm; column temperature 30 ° C, flow rate 1.0 ml / min. Elution conditions are shown in Table 9

[0086] Table 9 Mobile phase conditions

[0087]

[0088] Preparation of reference solution: Weigh 5 mg of Pulsatilloside A and 23-hydroxybetulinic acid respectively, accurately weigh, place in a 25 ml volumetric flask, add methanol to dissolve to the scale, filter, and take the filtrate to obtain the reference solution.

[0089] Preparation of test solution: Accurately measure 5.0 ml of Pulsatilla saponin reaction solution, place it in a 25 ml volumetric flask, add methanol to dissolve to the scale, filter, and take the filtrate to obtain the test solution.

[0090] Determination: Accurately aspirate 10μ1 of reference solution and sample solution and inject into liquid chromatograph for determination.

[0091] 2.1 Investigation of acid types

[0092] Take 3 portions of Pulsatilla saponin A3, 1 g each, weigh accurately, add 20 times 2% H2SO4 methanol solution, 20 times 2% HCl methanol solution, 20 times 2% H3PO4 methanol solution, heat and reflux at 50°C for 1 hour, add dilute sodium hydroxide solution to adjust the pH to 6-7. Shake well, and test according to the content determination method. The results are shown in Table 10. Table 10 Acid type investigation results

[0093]

[0094] According to the results of the acid type investigation, the concentration of Pulsatilloside A was used as an indicator to perform hydrolysis under different types of acid solutions. The results showed that all three acids could convert Pulsatilloside A3 into Pulsatilloside A, among which 2% H2SO4 methanol solution had the highest concentration of Pulsatilloside A, followed by 2% HCl methanol solution, and 2% H3PO4 methanol solution had the worst hydrolysis effect. Therefore, H2SO4 was selected for the experiment.

[0095] 2.2 Investigation of acid hydrolysis temperature

[0096] Take 3 portions of Pulsatilla saponin A3, 1 g each, weigh accurately, add 20 times 2% H2SO4 methanol solution, heat and reflux at 50℃, 60℃, and 70℃ for 1 hour, add dilute sodium hydroxide solution to adjust the pH to 6-7. Shake well and test according to the content determination method. The results are shown in Table 11

[0097] Table 11 Hydrolysis temperature investigation results

[0098]

[0099] According to the results of the acid hydrolysis temperature investigation, the concentration of Pulsatilloside A was used as an indicator to perform hydrolysis at different temperatures. The results showed that the concentration of Pulsatilloside A was the highest when the hydrolysis temperature was 60°C, and the concentration of Pulsatilloside A decreased at 70°C, while the concentration of 23-hydroxybetulinic acid increased relatively, indicating that as the temperature increased, the reaction continued in the positive direction, and the reaction temperature needed to be strictly controlled. Therefore, 60°C was selected for the experiment.

[0100] 2.3 Acid concentration investigation

[0101] Take 3 portions of Pulsatilla saponin A3, 1 g each, weigh accurately, add 20 times 1% H2SO4 methanol solution, 2% H2SO4 methanol solution, 3% H2SO4 methanol solution respectively, heat and reflux at 60℃ for 1h, add dilute sodium hydroxide solution to adjust pH to 6-7. Shake well and test according to the content determination method. The results are shown in Table 12

[0102] Table 12 Acid concentration investigation results

[0103]

[0104] According to the results of the acid concentration investigation, the concentration of Pulsatilloside A was used as an indicator to perform hydrolysis under different acid concentrations. The results showed that the concentration of Pulsatilloside A was the highest when the acid concentration was 2%, followed by 3%, and the lowest when 1%. This indicates that when the acid concentration is low, the reaction efficiency is low; when the acid concentration is too high, the reaction is excessive and the reaction continues to proceed toward 23-hydroxybetulinic acid; when the acid concentration is 2%, the concentrations of Pulsatilloside A and 23-hydroxybetulinic acid reach equilibrium, and the concentration of Pulsatilloside A is the highest at this time. Therefore, 2% H2SO4 was selected for the experiment.

[0105] 2.4 Investigation of material-liquid ratio

[0106] Take 3 portions of Pulsatilla saponin A3, 1 g each, add 2% H2SO4 methanol solution at a solid-liquid ratio of 1:40, 1:20, and 1:10, respectively, heat and reflux at 60°C for 1 hour, add dilute sodium hydroxide solution to adjust the pH to 6-7. Shake well and test according to the content determination method. The results are shown in Table 13

[0107] Table 13 Results of investigation on material-liquid ratio

[0108]

[0109] According to the results of the investigation of the solid-liquid ratio, the amount of Pulsatilloside A was used as an indicator, and hydrolysis was carried out at different solid-liquid ratios. The results showed that the amount of Pulsatilloside A generated at a solid-liquid ratio of 1:20 and 1:40 was similar, and the amount of Pulsatilloside A generated at 1:10 was second. This shows that the reaction efficiency is higher when the solid-liquid ratio is 1:20-40.

[0110] 2.5 Acid hydrolysis time investigation

[0111] Take 3 portions of Pulsatilla saponin A3, 1 g each, weigh accurately, add 20 times 2% H2SO4 methanol solution, heat and reflux at 60°C for 0.5h, 1h, and 1.5h, respectively, add dilute sodium hydroxide solution to adjust the pH to 6-7. Shake well and test according to the content determination method. The results are shown in Table 14

[0112] Table 14 Acid hydrolysis time investigation results

[0113]

[0114] According to the results of the acid hydrolysis time investigation, the concentration of Pulsatilloside A was used as an indicator, and the hydrolysis time was different. The results showed that with the increase of time, the concentration of Pulsatilloside A gradually decreased, and the concentration of 23-hydroxybetulinic acid gradually increased. This shows that with the increase of time, the reaction continues to proceed toward 23-hydroxybetulinic acid. Therefore, the reaction time of 0.5h was selected for the experiment.

[0115] According to the results of the acid hydrolysis single factor test, the conditions for the reaction of Pulsatilloside A3 to Pulsatilloside A were proposed as follows: solid-liquid ratio 1:20-40, adding 20 times 2% H2SO4 methanol solution, reacting at 60°C for 0.5h, and adjusting the pH to 6-7 with dilute sodium hydroxide.

[0116] 2.6 Acid hydrolysis process validation

[0117] Take 3 portions of Pulsatilla saponin A3, 5 g each, weigh accurately, add 20 times 2% H2SO4 methanol solution respectively, heat and reflux at 60℃ for 0.5h, then add dilute sodium hydroxide to adjust the pH to 6-7. Shake well and test according to the content determination method. The results are shown in Table 15

[0118] Table 15 Results of acid hydrolysis process verification investigation

[0119]

[0120] According to the process validation results, Pulsatilloside A concentration was used as an indicator and hydrolysis was carried out under the single-factor optimal conditions. The results showed that the concentrations of Pulsatilloside A in the three batches were basically the same.

[0121] 3. Purification of Pulsatilloside A

[0122] Take the acid hydrolysis process verification sample, use C-18 filler as the stationary phase, water as mobile phase A, methanol as mobile phase B, the detection wavelength is 203nm, perform high pressure preparation according to the gradient in the table below, collect the sample segment, and dry it.

[0123] Table 16 High pressure preparation mobile phase ratio

[0124]

[0125] 4. Pulsatilloside A structure identification

[0126] The structure was identified by H NMR, C NMR and mass spectrometry, and the purity was analyzed by HPLC. Figure 1 .

[0127] According to the results of H NMR, C NMR and mass spectrometry, the compound was identified to be consistent with the structure of Pulsatilloside A.

[0128] Example 3 Detection method of Pulsatilloside A of the present invention

[0129] HPLC detection was adopted, and the chromatographic conditions were:

[0130] Octadecylsilane bonded silica gel was used as filler (Agilent Extend-C18, 4.6 mm × 250 mm, 5 μm); water (A) and acetonitrile (B) were used as mobile phases; the detection wavelength was 201 nm; the column temperature was 30°C, the flow rate was 1.0 ml / min, and the elution conditions were shown in the following table:

[0131] Mobile phase conditions

[0132]

[0133] Preparation of reference solution: Take appropriate amount of Pulsatilloside A3, Pulsatilloside A, and 23-hydroxybetulinic acid reference substances, weigh them accurately, place them in a brown volumetric flask, and add methanol to make solutions containing 0.5 mg, 0.35 mg, and 0.6 mg per 1 ml.

[0134] Preparation of the test solution: Take an appropriate amount of Pulsatilloside A reaction solid, weigh it accurately, place it in a brown volumetric flask, and add methanol to the scale.

[0135] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0136] HPLC chromatogram Figure 2 ; The relative retention time of Pulsatilloside A in the spectrum is 22.63min.

Claims

1. A method for synthesizing Pulsatilloside A, characterized in that: It is prepared from pulsatilla saponin B4 as the starting material, and its synthetic route is:

2. The method for synthesizing Pulsatilloside A according to claim 1, characterized in that: It includes the following steps: a. Weigh 1 part of Pulsatilla saponin B4, add 10-20 parts of 2.5%-10% alkaline solution, heat and reflux at 50-70°C for 0.5-1.5h, add dilute sulfuric acid to adjust the pH to 6-7, and obtain Pulsatilla saponin A3; the alkaline solution is NaOH aqueous solution, KOH aqueous solution, ammonia aqueous solution; b. Purification of Pulsatilla saponin A3; c. Take the purified Pulsatilla saponin A3, add 10-40 times 1%-3% acid solution, heat and reflux at 50-70°C for 0.5-1.5h, add dilute sodium hydroxide solution to adjust the pH to 6-7; the acid solution is H2SO4 methanol solution, HCl methanol solution, H3PO4 methanol solution; d. Purification: Take the mixture prepared in step c, use C-18 filler as the stationary phase, water as mobile phase A, methanol as mobile phase B, and the detection wavelength is 201 nm. Perform high pressure preparation according to the gradient in the table below, collect the sample segment, and dry it. High pressure preparation mobile phase ratio 3. The method for synthesizing Pulsatilloside A according to claim 2, characterized in that: The alkaline solution described in step a is a NaOH or KOH aqueous solution with a concentration of 5%, and the material-liquid ratio is 41 parts of Pulsatilla saponin B: 20 ​​parts of alkaline solution; the heating reflux temperature is 60°C-70°C, and the reflux time is 1-1.5h.

4. The method for synthesizing Pulsatilloside A according to claim 2, characterized in that: The acid solution is H2SO4 methanol solution with a concentration of 2%; the material-liquid ratio is 1:20-40; the hydrolysis temperature is 60°C and the time is 0.5h.

5. A method for detecting Pulsatilloside A, characterized in that: It is detected by HPLC, and the chromatographic conditions are: Octadecylsilane bonded silica gel was used as filler (Agilent Extend-C18, 4.6 mm × 250 mm, 5 μm); water (A) and acetonitrile (B) were used as mobile phases; the detection wavelength was 201 nm; the column temperature was 30°C, the flow rate was 1.0 ml / min, and the elution conditions were shown in the following table: Mobile phase conditions 6. The method for detecting Pulsatilloside A according to claim 5, characterized in that: It includes the following steps: a. Preparation of reference solution: Take Pulsatilloside A3, Pulsatilloside A, and 23-hydroxybetulinic acid reference substances, accurately weigh them, place them in a brown volumetric bottle, and add methanol to make solutions containing 0.5 mg, 0.35 mg, and 0.6 mg per 1 ml. b. Preparation of test solution Take the Pulsatilloside A reaction solid, weigh it accurately, put it in a brown volumetric flask, add methanol to the mark, and obtain; c. Determination method: Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into a liquid chromatograph, and measure according to the chromatographic conditions described in claim 5 to obtain the product.

7. The method for detecting Pulsatilloside A according to claim 5 or 6, characterized in that: The relative retention time of Pulsatilloside A is 22.63±0.52min.

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